Current testing programs for pesticides adequately capture endocrine activity and adversity for protection of vertebrate wildlife.

The toxicity and ecotoxicity of pesticide active ingredients are evaluated by a number of standardized test methods using vertebrate animals. These standard test methods are required under various regulatory programs for the registration of pesticides. Over the past two decades, additional test methods have been developed with endpoints that are responsive to endocrine activity and subsequent adverse effects. This article examines the available test methods and their endpoints that are relevant to an assessment of endocrine-disrupting properties of pesticides. Furthermore, the article highlights how weight-of-evidence approaches should be applied to determine whether an adverse response in (eco)toxicity tests is caused by an endocrine mechanism of action. The large number of endpoints in the current testing paradigms for pesticides make it unlikely that endocrine activity and adversity is being overlooked. Integr Environ Assess Manag 2023;19:1089-1109. © 2023 Bayer CropScience and The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

The Extended Amphibian Metamorphosis Assay: A Thyroid-Specific and Less Animal-Intensive Alternative to the Larval Amphibian Growth and Development Assay.

The amphibian metamorphosis assay (AMA; US Environmental Protection Agency [USEPA] test guideline 890.1100 and Organisation for Economic Co-Operation and Development test guideline 231) has been used for more than a decade to assess the potential thyroid-mediated endocrine activity of chemicals. In 2013, in the context of the Endocrine Disruptor Screening Program of the USEPA, a Scientific Advisory Panel reviewed the results from 18 studies and recommended changes to the AMA test guideline, including a modification to a fixed-stage design rather than a fixed-time (i.e., 21-d) design. We describe an extended test design for the AMA (or EAMA) that includes thyroid histopathology and time to metamorphosis (Nieuwkoop-Faber [NF] stage 62), to address both the issues with the fixed-time design and the specific question of thyroid-mediated adversity in a shorter assay than the larval amphibian growth and development assay (LAGDA; Organisation for Economic Co-Operation and Development test guideline 241), using fewer animals and resources. A demonstration study was conducted with the EAMA (up to NF stage 58) using sodium perchlorate. Data analyses and interpretation of the fixed-stage design of the EAMA are more straightforward than the fixed-time design because the fixed-stage design avoids confounded morphometric measurements and thyroid histopathology caused by varying developmental stages at test termination. It also results in greater statistical power to detect metamorphic delays than the fixed-time design. By preferentially extending the AMA to NF stage 62, suitable data can be produced to evaluate thyroid-mediated adversity and preclude the need to perform a LAGDA for thyroid mode of action analysis. The LAGDA remains of further interest should investigations of longer term effects related to sexual development modulated though the hypothalamus-pituitary-gonadal axis be necessary. However, reproduction assessment or life cycle testing is currently not addressed in the LAGDA study design. This is better addressed by higher tier studies in fish, which should then include specific thyroid-related endpoints. Environ Toxicol Chem 2021;40:2135-2144. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Critical Review of Read-Across Potential in Testing for Endocrine-Related Effects in Vertebrate Ecological Receptors.

Recent regulatory testing programs have been designed to evaluate whether a chemical has the potential to interact with the endocrine system and could cause adverse effects. Some endocrine pathways are highly conserved among vertebrates, providing a potential to extrapolate data generated for one vertebrate taxonomic group to others (i.e., biological read-across). To assess the potential for biological read-across, we reviewed tools and approaches that support species extrapolation for fish, amphibians, birds, and reptiles. For each of the estrogen, androgen, thyroid, and steroidogenesis (EATS) pathways, we considered the pathway conservation across species and the responses of endocrine-sensitive endpoints. The available data show a high degree of confidence in the conservation of the hypothalamus-pituitary-gonadal axis between fish and mammals and the hypothalamus-pituitary-thyroid axis between amphibians and mammals. Comparatively, there is less empirical evidence for the conservation of other EATS pathways between other taxonomic groups, but this may be due to limited data. Although more information on sensitive pathways and endpoints would be useful, current developments in the use of molecular target sequencing similarity tools and thoughtful application of the adverse outcome pathway concept show promise for further advancement of read-across approaches for testing EATS pathways in vertebrate ecological receptors. Environ Toxicol Chem 2020;39:739-753. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Scientific integrity issues in Environmental Toxicology and Chemistry: Improving research reproducibility, credibility, and transparency.

High-profile reports of detrimental scientific practices leading to retractions in the scientific literature contribute to lack of trust in scientific experts. Although the bulk of these have been in the literature of other disciplines, environmental toxicology and chemistry are not free from problems. While we believe that egregious misconduct such as fraud, fabrication of data, or plagiarism is rare, scientific integrity is much broader than the absence of misconduct. We are more concerned with more commonly encountered and nuanced issues such as poor reliability and bias. We review a range of topics including conflicts of interests, competing interests, some particularly challenging situations, reproducibility, bias, and other attributes of ecotoxicological studies that enhance or detract from scientific credibility. Our vision of scientific integrity encourages a self-correcting culture that promotes scientific rigor, relevant reproducible research, transparency in competing interests, methods and results, and education. Integr Environ Assess Manag 2019;00:000-000. © 2019 SETAC.

Relevance weighting of tier 1 endocrine screening endpoints by rank order.

Weight of evidence (WoE) approaches are recommended for interpreting various toxicological data, but few systematic and transparent procedures exist. A hypothesis-based WoE framework was recently published focusing on the U.S. EPA's Tier 1 Endocrine Screening Battery (ESB) as an example. The framework recommends weighting each experimental endpoint according to its relevance for deciding eight hypotheses addressed by the ESB. Here we present detailed rationale for weighting the ESB endpoints according to three rank ordered categories and an interpretive process for using the rankings to reach WoE determinations. Rank 1 was assigned to in vivo endpoints that characterize the fundamental physiological actions for androgen, estrogen, and thyroid activities. Rank 1 endpoints are specific and sensitive for the hypothesis, interpretable without ancillary data, and rarely confounded by artifacts or nonspecific activity. Rank 2 endpoints are specific and interpretable for the hypothesis but less informative than Rank 1, often due to oversensitivity, inclusion of narrowly context-dependent components of the hormonal system (e.g., in vitro endpoints), or confounding by nonspecific activity. Rank 3 endpoints are relevant for the hypothesis but only corroborative of Ranks 1 and 2 endpoints. Rank 3 includes many apical in vivo endpoints that can be affected by systemic toxicity and nonhormonal activity. Although these relevance weight rankings (WREL ) necessarily involve professional judgment, their a priori derivation enhances transparency and renders WoE determinations amenable to methodological scrutiny according to basic scientific premises, characteristics that cannot be assured by processes in which the rationale for decisions is provided post hoc.

Lessons learned, challenges, and opportunities: the U.S. Endocrine Disruptor Screening Program.

In 1996, the U.S. Congress passed the Food Quality Protection Act and amended the Safe Drinking Water Act (SDWA) requiring the U.S. Environmental Protection Agency (EPA) to implement a screening program to investigate the potential of pesticide chemicals and drinking water contaminants to adversely affect endocrine pathways. Consequently, the EPA launched the Endocrine Disruptor Screening Program (EDSP) to develop and validate estrogen, androgen, and thyroid (EAT) pathway screening assays and to produce standardized and harmonized test guidelines for regulatory application. In 2009, the EPA issued the first set of test orders for EDSP screening and a total of 50 pesticide actives and 2 inert ingredients have been evaluated using the battery of EDSP Tier 1 screening assays (i.e., five in vitro assays and six in vivo assays). To provide a framework for retrospective analysis of the data generated and to collect the insight of multiple stakeholders involved in the testing, more than 240 scientists from government, industry, academia, and non-profit organizations recently participated in a workshop titled "Lessons Learned, Challenges, and Opportunities: The U.S. Endocrine Disruptor Screening Program." The workshop focused on the science and experience to date and was organized into three focal sessions: (a) Performance of the EDSP Tier 1 Screening Assays for Estrogen, Androgen, and Thyroid Pathways; (b) Practical Applications of Tier 1 Data; and (c) Indications and Opportunities for Future Endocrine Testing. A number of key learnings and recommendations related to future EDSP evaluations emanated from the collective sessions.

Hypothesis-driven weight of evidence framework for evaluating data within the US EPA's Endocrine Disruptor Screening Program.

"Weight of Evidence" (WoE) approaches are often used to critically examine, prioritize, and integrate results from different types of studies to reach general conclusions. For assessing hormonally active agents, WoE evaluations are necessary to assess screening assays that identify potential interactions with components of the endocrine system, long-term reproductive and developmental toxicity tests that define adverse effects, mode of action studies aimed at identifying toxicological pathways underlying adverse effects, and toxicity, exposure and pharmacokinetic data to characterize potential risks. We describe a hypothesis-driven WoE approach for hormonally active agents and illustrate the approach by constructing hypotheses for testing the premise that a substance interacts as an agonist or antagonist with components of estrogen, androgen, or thyroid pathways or with components of the aromatase or steroidogenic enzyme systems for evaluating data within the US EPA's Endocrine Disruptor Screening Program. Published recommendations are used to evaluate data validity for testing each hypothesis and quantitative weightings are proposed to reflect two data parameters. Relevance weightings should be derived for each endpoint to reflect the degree to which it probes each specific hypothesis. Response weightings should be derived based on assay results from the test substance compared to the range of responses produced in the assay by the appropriate prototype hormone and positive and negative controls. Overall WoE scores should be derived based on response and relevance weightings and a WoE narrative developed to clearly describe the final determinations.

Acute and chronic toxicity testing of bisphenol A with aquatic invertebrates and plants.

Bisphenol A (BPA, 4,4'-isopropylidine diphenol) is a commercially important chemical used primarily as an intermediate in the production of polycarbonate plastic and epoxy resins. Extensive effect data are currently available, including long-term studies with BPA on fish, amphibians, crustaceans, and mollusks. The aim of this study was to perform additional tests with a number of aquatic invertebrates and an aquatic plant. These studies include acute tests with the midge (Chironomus tentans) and the snail (Marisa cornuarietis), and chronic studies with rotifers (Brachionus calyciflorus), amphipods (Hyalella azteca), and plants (Lemna gibba). The effect data on different aquatic invertebrate and plant species presented in this paper correspond well with the effect and no-effect concentrations (NOECs) available from invertebrate studies in the published literature and are within the range found for other aquatic species tested with BPA.

Sources of variability in plant toxicity testing.

Published literature is investigated regarding the response of plants to various substances to determine the sensitivity of agricultural plants versus other species, the similarity of effects seen at different taxonomic levels, sensitivity of plants growing outdoors versus in a greenhouse, and the sensitivity of different measurement endpoints. We find that agricultural species are not consistently more or less sensitive to the herbicides tested than non-crop species. Genus and family taxonomic groupings may show similar responses among species, but this similarity quickly decreases as the comparison progress between orders and classes. Results from field and greenhouse studies are less in agreement between studies than data from the other topics. Shoot length will be affected at concentrations lower than for other vegetative endpoints for most species tested for inorganic substances, but for organic substances root and shoot mass were more sensitive. Overall, there is no one species or endpoint that is consistently the most sensitive for all species or all chemicals in all soils, and differences in bioavailability among compounds may confound comparison of test results. Therefore, species sensitivity distributions, adjusted for bioavailability when possible, should be considered in order to better evaluate effects to non-target terrestrial plants.